Methods of Analysis of Allelic Imbalance

ABSTRACT

Methods are provided for identification of genes that are imprinted. In another embodiment methods are provided for identification and analysis of genes whose expression shows allelic imbalance. The expression products transcribed from genes that are present in the genome as two or more alleles may be distinguished by hybridization to an array designed to interrogate individual alleles. Genes whose transcription products are present in amounts that vary from expected are candidates for allelic imbalance, imprinting and imprinting errors.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/389,745, filed Jun. 17, 2002, the disclosure of which is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to determining the imprinting status of genes. Inone embodiment, the invention relates to identification of genes thatare present in two allelic forms and show differential expression of thedifferent alleles. The methods may be used to identify changes inimprinting status, to diagnose disease and to predict likelihood ofdisease. The present invention relates to the fields of molecularbiology and genetics.

BACKGROUND

Each mammalian cell carries two copies of each gene, one inherited fromthe mother (on the maternal chromosome) and one inherited from thefather (on the paternal chromosome). Most of the autosomal genes andX-linked genes in females are therefore biallelic i.e. both paternal andmaternal alleles of the gene are expressed and the information of bothcopies is actively used in protein synthesis. In males, sex-linked genesare generally monoallelic since there is one X and one Y chromosome.Only a few genes on the Y chromosome have functional homologs on the Xchromosome and are biallelic.

However, in humans and other mammals, monoallelic expression ofbiallelic genes has been demonstrated. These include genes on theinactive X-chromosome, genes encoding IL-2, IL-4, PAX-5, subunits ofolfactory and lymphocyte receptors and imprinted genes. Allelicexclusion can result from two different mechanisms. The first mechanismis independent of the parental origin. One allele is randomly repressedand the pattern of allelic exclusion is transmitted stably to thedaughter cells. This allelic exclusion can be due to X-chromosomeinactivation, to programmed DNA rearrangement (B and T cell receptor inlymphocytes) or to other unknown mechanisms. The second mechanism,called genomic imprinting, is the result of a mark or imprint carried bya region of the chromosome and that reflects the parental origin.Imprinted genes in the mammalian genome are the genes for which one ofthe parental alleles is repressed whereas the other one is transcribedand expressed. Many imprinted genes are located in clusters and areassociated with CpG-rich regions called CpG islands that are methylateduniquely on a specific parental chromosome (Razin A. and Cedar H. (1994)Cell, 77:473-476; Constancia M et al. (1998) 8:881-900, Reik W. andWalter J. (2001) Nature Rev. Genet., 2:21-32 incorporated in theirentity by reference for all purposes).

About sixty imprinted genes have been discovered in the mouse. Anestimate of one to two hundred imprinted genes has been proposed basedon mouse models (Barlow D. P. (1995) Science, 270: 1610-1613; Morison I.M. et al. (2001), Nucl. Acids Res., 29:275-276 each of which isincorporated herein by reference in its entirety; databases available athttp://www.otago.ac.nz/IGC and http://www.geneimprint.com).

Imprinted genes tend to occur in clusters in both the human and mousegenomes (Reik W. and Walter J. (1998) Curr. Opin. Genet., 8:154-164),which is incorporated herein by reference in its entirety. For example,in humans, two chromosomal regions (11p15.5 and 15q11-q13) harbor morethan one imprinted gene. Some imprinted genes, such as Igf2(Insulin-like growth factor type 2) and H19 (a non-coding RNA involvedin silencing Igf2 expression) are located in imprinted clusters of genesthat show coordinate regulation.

Imprinted genes can show monoallelic expression in some tissues andbiallelic expression in others. For example, Igf2 is imprinted in mosttissues but is biallelic in brain, liver and several other tissues.Monoallelic expression or disruption of monoallelic expression of somegenes can lead to a disease phenotype. For instance, imprinting is afactor in an increasing number of genetic diseases such as Prader-Willisyndrome, Angelman syndrome, and Beckwith-Wiedmann syndrome. Imprintedgenes and imprinting mechanisms are therefore important in human birthdefects, cancer and in some neurological and psychiatric disorders (forreview, see Falls G. J. et al. (1999) Am. J. Path., 154:635-647).

Monoallelic expression of some genes that are present in two copies isrequired for normal development and viability. For example, humanfemales have two copies of the X chromosome while males have a single Xchromosome. However, females have effectively only a single copy of theX chromosome due to inactivation of one copy of the X chromosome in eachcell. The inactive copy is known as a Barr body and inactivation isrequired for normal development. Inactivation of the X chromosome israndom, resulting in mosaicism, meaning that in some cells the paternalcopy of the X chromosome is inactivated and in some cells the maternalcopy is inactivated. For genes that are present in a different allelicform on the paternal and maternal X chromosomes this results inexpression of one allele in some cells and the other allele in othercells.

SUMMARY OF THE INVENTION

In one aspect methods are provided for identifying at least oneheterozygous gene showing monoallelic expression in an individual. Themethod includes the steps of providing a genomic DNA sample from theindividual; providing a nucleic acid array comprising probes designed tointerrogate a plurality of polymorphisms; hybridizing the genomic sampleto a first copy of the array; generating a hybridization patternresulting from the hybridization; analyzing the hybridization pattern todetermine the identity of the alleles present for at least onepolymorphism in the plurality of polymorphisms; identifying at least onepolymorphism in the plurality of polymorphisms that is heterozygous inthe individual; isolating an RNA sample from the individual; hybridizinga nucleic acid sample derived from the RNA sample to the same array orto a second copy of the array and generating a hybridization pattern;and identifying at least one polymorphism in the plurality ofpolymorphisms that is heterozygous in the genome and homozygous in theRNA sample.

The polymorphisms may be single nucleotide polymorphisms. In someembodiments the polymorphisms are associated with a phenotype, forexample a disease such as cancer or a neurological disorder like bipolardisorder or schizophrenia.

Monoallelic expression may be the result of imprinting and the parentalorigin of the expressed allele may be determined by establishing if theexpressed allele is present in the maternal or paternal genome. Someimprinted genes express only the maternal copy of the gene and otherimprinted genes express only the paternal copy of the gene. Imprintedgenes may encode, for example, a lymphoid-specific factor, a subunit ofan olfactory receptor, a subunit of a T cell receptor or a subunit of animmunoglobulin.

In many aspects, the samples derived from the genomic DNA and the RNAare differentially labeled. This allows both samples to be hybridized tothe same array. Hybridization may be sequential or simultaneous wherethe sample may be mixed before or on the array. The differential labelsallow separation of the hybridization patterns on the array. The nucleicacid samples that are hybridized may be genomic DNA or transcribed RNAthat has been directly labeled, but in many embodiments the hybridizedsample has been derived from the genomic DNA or transcribed RNA sampleby, for example, amplification.

In one aspect, methods are provided for determining if the imprinting ofa specific gene is tissue specific. The methods comprise the steps ofidentifying at least one heterozygous SNP in an imprinted gene in anindividual; providing a nucleic acid array comprising probes designed tointerrogate the SNP; isolating an RNA sample from each of a plurality ofdifferent tissue samples from the individual; hybridizing the RNA sampleor a nucleic acid derived from the RNA sample from each tissue sample toan array to generate a hybridization pattern for each tissue sample; andanalyzing the hybridization patterns to determine if the gene showstissue specific imprinting.

In one aspect, methods are provided for determining if the imprinting ofa specific gene is cell specific by comparing the expression of the genein different cells of an individual. At least one heterozygous SNP onthe DNA of an imprinted gene is identified; an RNA sample is isolatedfrom a cell in which the gene is imprinted and from at least onedifferent cell type; the RNA samples or a nucleic acid derived from theRNA samples are hybridized to a genotyping array and the hybridizationpattern is analyzed to determine if the RNA is homozygous in both of theRNA samples. The RNAs from the different samples may be differentiallylabeled and hybridized to the same array either simultaneously orsequentially or they can be labeled with the same label and hybridizedto separate arrays.

In one aspect, methods are provided for determining if the imprinting ofa specific gene is species specific. The expression of the imprintedgene is analyzed in samples of the same tissue type from differentspecies.

In one aspect, methods are provided for determining if the imprintingstatus of an imprinted gene is polymorphic in a population. Theexpression of the imprinted gene is compared between differentindividuals in a population. If the gene is imprinted in all of theindividuals of the population it is not polymorphic in the population.If some individuals show imprinting of the gene, but other individualsdo not, then the gene is identified as having a polymorphic imprintingstatus in that population.

In one aspect, imprinting is used to determine if a patient has anincreased risk of developing a disease due to loss of imprinting.Heterozygous imprinted genes are identified in healthy individuals andthese genes are analyzed in sample individuals to detect heterozygousgenes that are imprinted in healthy individual but not imprinted in thepatient.

In another aspect, a method is provided for determining if apreimplantation embryo has an increased risk of developing a disease dueto abnormal imprinting.

In another aspect, a method is provided for identifying novel imprintedgenes.

In another aspect, a method is provided for establishing a genome-wideimprinting chromosomal map. Genes that are heterozygous in the genomeand homozygous in the expressed RNA are identified by hybridizingnucleic acid from genomic DNA and from transcribed RNA to an array thatinterrogates a plurality of SNPs. Chromosomal regions that carry two ormore imprinted genes are identified on a genomic map. This mapping maybe done, for example, for the human genome, for a specific tissue type,or for a specific developmental stage or stages. A genome-wideimprinting map may be used, for example, to identify genomic regionsassociated with disease.

In one aspect, methods are provided for assessing the genomic imprintingstatus of a cloned embryo. Cloning and nuclear cell transfer may resultin abnormal imprinting and may result in abnormal phenotypes in clonedorganisms. Imprinting in cloned individual may be compared withimprinting in normal individuals.

In one aspect, a method is provided for assessing the genomic imprintingstatus of genes from a transplantation tissue or cell.

In one aspect, a method is provided for identifying an agent that maycause imprinting deregulation in an individual, tissue or cell. Theindividual, tissue or cell is treated with the agent and imprinting iscompared between treated and untreated samples.

DETAILED DESCRIPTION OF THE INVENTION I. General

The present invention has many preferred embodiments and relies on manypatents, applications and other references for details known to those ofthe art. Therefore, when a patent, application, or other reference iscited or repeated below, it should be understood that it is incorporatedby reference in its entirety for all purposes as well as for theproposition that is recited.

As used in this application, the singular form “a,” “an,” and “the”include plural references unless the context clearly dictates otherwise.For example, the term “an agent” includes a plurality of agents,including mixtures thereof.

An individual is not limited to a human being but may also be otherorganisms including but not limited to mammals, plants, bacteria, orcells derived from any of the above.

Throughout this disclosure, various aspects of this invention can bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and immunology, which arewithin the skill of the art. Such conventional techniques includepolymer array synthesis, hybridization, ligation, and detection ofhybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the example herein below. However,other equivalent conventional procedures can, of course, also be used.Such conventional techniques and descriptions can be found in standardlaboratory manuals such as Genome Analysis: A Laboratory Manual Series(Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A LaboratoryManual, PCR Primer: A Laboratory Manual, and Molecular Cloning: ALaboratory Manual (all from Cold Spring Harbor Laboratory Press),Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, New York, Gait,“Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press,London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry3^(rd) Ed., W. H. Freeman Pub., New York, N.Y. and Berg et al. (2002)Biochemistry, 5^(th) Ed., W. H. Freeman Pub., New York, N.Y., all ofwhich are herein incorporated in their entirety by reference for allpurposes.

The present invention can employ solid substrates, including arrays insome preferred embodiments. Methods and techniques applicable to polymer(including protein) array synthesis have been described in U.S. Ser. No.09/536,841, WO 00/58516, U.S. Pat. Nos. 5,143,854, 5,242,974, 5,252,743,5,324,633, 5,384,261, 5,405,783, 5,424,186, 5,451,683, 5,482,867,5,491,074, 5,527,681, 5,550,215, 5,571,639, 5,578,832, 5,593,839,5,599,695, 5,624,711, 5,631,734, 5,795,716, 5,831,070, 5,837,832,5,856,101, 5,858,659, 5,936,324, 5,968,740, 5,974,164, 5,981,185,5,981,956, 6,025,601, 6,033,860, 6,040,193, 6,090,555, 6,136,269,6,269,846 and 6,428,752, in PCT Applications Nos. PCT/US99/00730(International Publication Number WO 99/36760) and PCT/US01/04285, whichare all incorporated herein by reference in their entirety for allpurposes.

Patents that describe synthesis techniques in specific embodimentsinclude U.S. Pat. Nos. 5,412,087, 6,147,205, 6,262,216, 6,310,189,5,889,165, and 5,959,098. Nucleic acid arrays are described in many ofthe above patents, but the same techniques are applied to polypeptidearrays.

Nucleic acid arrays that are useful in the present invention includethose that are commercially available from Affymetrix (Santa Clara,Calif.) under the brand name GeneChip®. Example arrays are shown on thewebsite at affymetrix.com.

The present invention also contemplates many uses for polymers attachedto solid substrates. These uses include gene expression monitoring,profiling, library screening, genotyping and diagnostics. Geneexpression monitoring and profiling methods can be shown in U.S. Pat.Nos. 5,800,992, 6,013,449, 6,020,135, 6,033,860, 6,040,138, 6,177,248and 6,309,822. Genotyping and uses therefore are shown in U.S. Ser. No.60/319,253, 10/013,598, and U.S. Pat. Nos. 5,856,092, 6,300,063,5,858,659, 6,284,460, 6,361,947, 6,368,799 and 6,333,179. Other uses areembodied in U.S. Pat. Nos. 5,871,928, 5,902,723, 6,045,996, 5,541,061,and 6,197,506.

The present invention also contemplates sample preparation methods incertain preferred embodiments. Prior to or concurrent with genotyping,the genomic sample may be amplified by a variety of mechanisms, some ofwhich may employ PCR. See, e.g., PCR Technology: Principles andApplications for DNA Amplification (Ed. H. A. Erlich, Freeman Press, NY,N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (Eds.Innis, et al., Academic Press, San Diego, Calif., 1990); Mattila et al.,Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods andApplications 1, 17 (1991); PCR (Eds. McPherson et al., IRL Press,Oxford); and U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,1594,965,188,and 5,333,675, and each of which is incorporated herein byreference in their entireties for all purposes. The sample may beamplified on the array. See, for example, U.S. Pat. No 6,300,070 andU.S. patent application Ser. No. 09/513,300, which are incorporatedherein by reference.

Other suitable amplification methods include the ligase chain reaction(LCR) (e.g., Wu and Wallace, Genomics 4, 560 (1989), Landegren et al.,Science 241, 1077 (1988) and Barringer et al. Gene 89:117 (1990)),transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86,1173 (1989) and WO88/10315), selective amplification of targetpolynucleotide sequences (U.S. Pat. No. 6,410,276), consensus sequenceprimed polymerase chain reaction (CP-PCR) (U.S. Pat. No. 4,437,975),arbitrarily primed polymerase chain reaction (AP-PCR) (U.S. Pat. No.5,413,909, 5,861,245), self-sustained sequence replication (Guatelli etal., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990) and WO90/06995) andnucleic acid based sequence amplification (NABSA). (See, U.S. Pat. Nos.5,409,818, 5,554,517, and 6,063,603, each of which is incorporatedherein by reference). The latter two amplification methods involveisothermal reactions based on isothermal transcription, which produceboth single stranded RNA (ssRNA) and double stranded DNA (dsDNA) as theamplification products in a ratio of about 30 or 100 to 1, respectively.Other amplification methods that may be used are described in, U.S. Pat.Nos. 5,242,794, 5,494,810, 4,988,617 and in U.S. Ser. No. 09/854,317,each of which is incorporated herein by reference.

Additional methods of sample preparation and techniques for reducing thecomplexity of a nucleic sample are described in Dong et al., GenomeResearch 11, 1418 (2001), in U.S. Pat. No. 6,361,947, 6,391,592 and U.S.Patent application Ser. Nos. 09/916,135, Ser. No. 09/920,491, Ser. No.09/910,292, and Ser. No. 10/013,598.

Methods for conducting polynucleotide hybridization assays have beenwell developed in the art. Hybridization assay procedures and conditionswill vary depending on the application and are selected in accordancewith the general binding methods known including those referred to in:Maniatis et al. Molecular Cloning: A Laboratory Manual (3^(rd) Ed. ColdSpring Harbor, N.Y., 2002); Berger and Kimmel Methods in Enzymology,Vol. 152, Guide to Molecular Cloning Techniques (Academic Press, Inc.,San Diego, Calif., 1987); Young and Davism, P.N.A.S, 80: 1194 (1983).Methods and apparatus for carrying out repeated and controlledhybridization reactions have been described in U.S. Pat. Nos. 5,871,928,5,874,219, 6,045,996 and 6,386,749, 6,391,623 each of which areincorporated herein by reference

The present invention also contemplates signal detection ofhybridization between ligands in certain preferred embodiments. See U.S.Pat. Nos. 5,143,854, 5,578,832; 5,631,734; 5,834,758; 5,936,324;5,981,956; 6,025,601; 6,141,096; 6,185,030; 6,201,639; 6,218,803; and6,225,625, in U.S. Patent application 60/364,731 and in PCT ApplicationPCT/US99/06097 (published as WO99/47964), each of which also is herebyincorporated by reference in its entirety for all purposes.

Methods and apparatus for signal detection and processing of intensitydata are disclosed in, for example, U.S. Pat. Nos. 5,143,854, 5,547,839,5,578,832, 5,631,734, 5,800,992, 5,834,758; 5,856,092, 5,902,723,5,936,324, 5,981,956, 6,025,601, 6,090,555, 6,141,096, 6,185,030,6,201,639; 6,218,803; and 6,225,625, in U.S. Patent application60/364,731 and in PCT Application PCT/US99/06097 (published asWO99/47964), each of which also is hereby incorporated by reference inits entirety for all purposes.

The practice of the present invention may also employ conventionalbiology methods, software and systems. Computer software products of theinvention typically include computer readable medium havingcomputer-executable instructions for performing the logic steps of themethod of the invention. Suitable computer readable medium includefloppy disk, CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM,magnetic tapes and etc. The computer executable instructions may bewritten in a suitable computer language or combination of severallanguages. Basic computational biology methods are described in, e.g.Setubal and Meidanis et al., Introduction to Computational BiologyMethods (PWS Publishing Company, Boston, 1997); Salzberg, Searles,Kasif, (Ed.), Computational Methods in Molecular Biology, (Elsevier,Amsterdam, 1998); Rashidi and Buehler, Bioinformatics Basics:Application in Biological Science and Medicine (CRC Press, London, 2000)and Ouelette and Bzevanis Bioinformatics: A Practical Guide for Analysisof Gene and Proteins (Wiley & Sons, Inc., 2^(nd) ed., 2001).

The present invention may also make use of various computer programproducts and software for a variety of purposes, such as probe design,management of data, analysis, and instrument operation. See, U.S. Pat.Nos. 5,593,839, 5,795,716, 5,733,729, 5,974,164, 6,066,454, 6,090,555,6,185,561, 6,188,783, 6,223,127, 6,229,911 and 6,308,170.

Additionally, the present invention may have preferred embodiments thatinclude methods for providing genetic information over networks such asthe Internet as shown in U.S. Patent applications Ser. No. 10/063,559,60/349,546, 60/376,003, 60/394,574, 60/403,381.

II. Definitions

An “individual” is not limited to a human being, but may also includeother organisms including but not limited to mammals, plants, bacteriaor cells derived from any of the above.

Nucleic acids according to the present invention may include any polymeror oligomer of pyrimidine and purine bases, preferably cytosine (C),thymine (T), and uracil (U), and adenine (A) and guanine (G),respectively. See Albert L. Lehninger, Principles of Biochemistry, at793-800 (Worth Pub. 1982) which is herein incorporated in its entiretyfor all purposes). Indeed, the present invention contemplates anydeoxyribonucleotide, ribonucleotide or peptide nucleic acid component,and any chemical variants thereof, such as methylated, hydroxymethylatedor glucosylated forms of these bases, and the like. The polymers oroligomers may be heterogeneous or homogeneous in composition, and may beisolated from naturally occurring sources or may be artificially orsynthetically produced. In addition, the nucleic acids may be DNA orRNA, or a mixture thereof, and may exist permanently or transitionallyin single-stranded or double-stranded form, including homoduplex,heteroduplex, and hybrid states. A second nucleic acid sample may bederived from a first nucleic acid sample by any method known in the art.For example, a genomic DNA sample may be amplified by PCR or any otheramplification method to generate a nucleic acid sample that is derivedfrom the genomic DNA sample. RNA or cDNA may be made from a genomic DNAsample using methods known in the art.

An “oligonucleotide” or “polynucleotide” is a nucleic acid ranging fromat least 2, preferably at least 8, and more preferably at least 20nucleotides in length or a compound that specifically hybridizes to apolynucleotide. Polynucleotides of the present invention includesequences of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) ormimetics thereof, which may be isolated from natural sources,recombinantly produced or artificially synthesized and mimetics thereof.A further example of a polynucleotide of the present invention may be apeptide nucleic acid (PNA) in which the constituent bases are joined bypeptides bonds rather than phosphodiester linkage, as described byNielsen et al., Science 254:1497-1500 (1991), Nielsen , Curr. Opin.Biotechnol., 10:71-75 (1999). The invention also encompasses situationsin which there is a nontraditional base pairing such as Hoogsteen basepairing which has been identified in certain tRNA molecules andpostulated to exist in a triple helix. “Polynucleotide” and“oligonucleotide” are used interchangeably in this application.

An “array” is an intentionally created collection of molecules which canbe prepared either synthetically or biosynthetically. The molecules inthe array can be identical or different from each other. The array canassume a variety of formats, e.g., libraries of soluble molecules;libraries of compounds tethered to resin beads, silica chips, or othersolid supports. Additionally, the term “array” is meant to include thoselibraries of nucleic acids which can be prepared by spotting nucleicacids of essentially any length (e.g., from 1 to about 1000 nucleotidemonomers in length) onto a substrate. The term “nucleic acid” as usedherein refers to a polymeric form of nucleotides of any length, eitherribonucleotides, deoxyribonucleotides or peptide nucleic acids (PNAs),that comprise purine and pyrimidine bases, or other natural, chemicallyor biochemically modified, non-natural, or derivatized nucleotide bases.The backbone of the polynucleotide can comprise sugars and phosphategroups, as may typically be found in RNA or DNA, or modified orsubstituted sugar or phosphate groups. A polynucleotide may comprisemodified nucleotides, such as methylated nucleotides and nucleotideanalogs. The sequence of nucleotides may be interrupted bynon-nucleotide components. Thus the terms nucleoside, nucleotide,deoxynucleoside and deoxynucleotide generally include analogs such asthose described herein. These analogs are those molecules having somestructural features in common with a naturally occurring nucleoside ornucleotide such that when incorporated into a nucleic acid oroligonucleotide sequence, they allow hybridization with a naturallyoccurring nucleic acid sequence in solution. Typically, these analogsare derived from naturally occurring nucleosides and nucleotides byreplacing and/or modifying the base, the ribose or the phosphodiestermoiety. The changes can be tailor made to stabilize or destabilizehybrid formation or enhance the specificity of hybridization with acomplementary nucleic acid sequence as desired.

“Solid support”, “support”, and “substrate” are used interchangeably andrefer to a material or group of materials having a rigid or semi-rigidsurface or surfaces. In many embodiments, at least one surface of thesolid support will be substantially flat, although in some embodimentsit may be desirable to physically separate synthesis regions fordifferent compounds with, for example, wells, raised regions, pins,etched trenches, or the like. According to other embodiments, the solidsupport(s) will take the form of beads, resins, gels, microspheres, orother geometric configurations.

Arrays may generally be produced using a variety of techniques, such asmechanical synthesis methods or light directed synthesis methods thatincorporate a combination of photolithographic methods and solid phasesynthesis methods. Techniques for the synthesis of these arrays usingmechanical synthesis methods are described in, e.g., U.S. Pat. Nos.5,384,261, and 6,040,193, which are incorporated herein by reference intheir entirety for all purposes. Although a planar array surface ispreferred, the array may be fabricated on a surface of virtually anyshape or even a multiplicity of surfaces. Arrays may be nucleic acids onbeads, gels, polymeric surfaces, fibers such as fiber optics, glass orany other appropriate substrate. (See U.S. Pat. Nos. 5,599,695,5,770,358, 5,789,162, 5,708,153, 6,040,193 and 5,800,992, which arehereby incorporated by reference in their entirety for all purposes.)

A “combinatorial synthesis strategy” is an ordered strategy for parallelsynthesis of diverse polymer sequences by sequential addition ofreagents which may be represented by a reactant matrix and a switchmatrix, the product of which is a product matrix. A reactant matrix isal column by m row matrix of the building blocks to be added. The switchmatrix is all or a subset of the binary numbers, preferably ordered,between l and m arranged in columns. A “binary strategy” is one in whichat least two successive steps illuminate a portion, often half, of aregion of interest on the substrate. In a binary synthesis strategy, allpossible compounds which can be formed from an ordered set of reactantsare formed. In most preferred embodiments, binary synthesis refers to asynthesis strategy which also factors a previous addition step. Forexample, a strategy in which a switch matrix for a masking strategyhalves regions that were previously illuminated, illuminating about halfof the previously illuminated region and protecting the remaining half(while also protecting about half of previously protected regions andilluminating about half of previously protected regions). It will berecognized that binary rounds may be interspersed with non-binary roundsand that only a portion of a substrate may be subjected to a binaryscheme. A combinatorial “masking” strategy is a synthesis which useslight or other spatially selective deprotecting or activating agents toremove protecting groups from materials for addition of other materialssuch as amino acids.

Arrays may be packaged in such a manner as to allow for diagnostic useor can be an all-inclusive device; e.g., U.S. Pat. Nos. 5,856,174 and5,922,591 incorporated in their entirety by reference for all purposes.Preferred arrays are commercially available from Affymetrix under thebrand name GeneChip® and are directed to a variety of purposes,including genotyping and gene expression monitoring for a variety ofeukaryotic and prokaryotic species. (See Affymetrix Inc., Santa Claraand their website at affymetrix.com.).

Complementary or substantially complementary refers to the hybridizationor base pairing between nucleotides or nucleic acids, such as, forinstance, between the two strands of a double stranded DNA molecule orbetween an oligonucleotide primer and a primer binding site on a singlestranded nucleic acid to be sequenced or amplified. Complementarynucleotides are, generally, A and T (or A and U), or C and G. Two singlestranded RNA or DNA molecules are the to be substantially complementarywhen the nucleotides of one strand, optimally aligned and compared andwith appropriate nucleotide insertions or deletions, pair with at leastabout 80% of the nucleotides of the other strand, usually at least about90% to 95%, and more preferably from about 98 to 100%. Alternatively,substantial complementarity exists when an RNA or DNA strand willhybridize under selective hybridization conditions to its complement.Typically, selective hybridization will occur when there is at leastabout 65% complementary over a stretch of at least 14 to 25 nucleotides,preferably at least about 75%, more preferably at least about 90%complementary. See, M. Kanehisa Nucleic Acids Res. 12:203 (1984),incorporated herein by reference.

The term “hybridization” refers to the process in which twosingle-stranded polynucleotides bind non-covalently to form a stabledouble-stranded polynucleotide; triple-stranded hybridization is alsotheoretically possible. The resulting (usually) double-strandedpolynucleotide is a “hybrid.” The proportion of the population ofpolynucleotides that forms stable hybrids is referred to herein as the“degree of hybridization”.

“Hybridization conditions” will typically include salt concentrations ofless than about 1M, more usually less than about 500 mM and preferablyless than about 200 mM. Hybridization temperatures can be as low as 5°C., but are typically greater than 22° C., more typically greater thanabout 30° C., and preferably in excess of about 37° C. Hybridizationsare usually performed under stringent conditions, i.e. conditions underwhich a probe will hybridize to its target subsequence. Stringentconditions are sequence-dependent and are different in differentcircumstances. Longer fragments may require higher hybridizationtemperatures for specific hybridization. As other factors may affect thestringency of hybridization, including base composition and length ofthe complementary strands, presence of organic solvents and extent ofbase mismatching, the combination of parameters is more important thanthe absolute measure of any one alone. Generally, stringent conditionsare selected to be about 5° C. lower than the thermal melting point (Tm)of the specific sequence at a defined ionic strength and pH. The Tm isthe temperature (under defined ionic strength, pH and nucleic acidcomposition) at which 50% of the probes complementary to the targetsequence hybridize to the target sequence at equilibrium. Typically,stringent conditions include salt concentration of at least 0.01 M to nomore than 1 M Na ion concentration (or other salts) at a pH 7.0 to 8.3and a temperature of at least 25° C. For example, conditions of 5X SSPE(750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4) and a temperature of25-30° C. are suitable for allele-specific probe hybridizations. Forstringent conditions, see for example, Sambrook, Fritsche and Maniatis.“Molecular Cloning A laboratory Manual” 2^(nd) Ed. Cold Spring HarborPress (1989) and Anderson “Nucleic Acid Hybridization” 1^(st) Ed., BIOSScientific Publishers Limited (1999), which are hereby incorporated byreference in its entirety for all purposes above.

“Hybridization probes” are oligonucleotides capable of binding in abase-specific manner to a complementary strand of nucleic acid. Suchprobes include peptide nucleic acids, as described in Nielsen et al.,Science 254:1497-1500 (1991), Nielsen Curr. Opin. Biotechnol., 10:71-75(1999) and other nucleic acid analogs and nucleic acid mimetics. See USPatent No. 6,156,501 filed Apr. 3, 1996.

“Hybridizing specifically to” refers to the binding, duplexing, orhybridizing of a molecule substantially to or only to a particularnucleotide sequence or sequences under stringent conditions when thatsequence is present in a complex mixture (e.g., total cellular) DNA orRNA.

A “probe” is a surface-immobilized molecule that can be recognized by aparticular target. Examples of probes that can be investigated by thisinvention include, but are not restricted to, agonists and antagonistsfor cell membrane receptors, toxins and venoms, viral epitopes, hormones(e.g., opioid peptides, steroids, etc.), hormone receptors, peptides,enzymes, enzyme substrates, cofactors, drugs, lectins, sugars,oligonucleotides, nucleic acids, oligosaccharides, proteins, andmonoclonal antibodies.

A “target” is a molecule that has an affinity for a given probe. Targetsmay be naturally-occurring or man-made molecules. Also, they can beemployed in their unaltered state or as aggregates with other species.Targets may be attached, covalently or noncovalently, to a bindingmember, either directly or via a specific binding substance. Examples oftargets which can be employed by this invention include, but are notrestricted to, antibodies, cell membrane receptors, monoclonalantibodies and antisera reactive with specific antigenic determinants(such as on viruses, cells or other materials), drugs, oligonucleotides,nucleic acids, peptides, cofactors, lectins, sugars, polysaccharides,cells, cellular membranes, and organelles. Targets are sometimesreferred to in the art as anti-probes. As the term targets is usedherein, no difference in meaning is intended. A “Probe Target Pair” isformed when two macromolecules have combined through molecularrecognition to form a complex.

An “isolated nucleic acid” is an object species invention that is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition). Preferably, anisolated nucleic acid comprises at least about 50, 80 or 90% (on a molarbasis) of all macromolecular species present. Most preferably, theobject species is purified to essential homogeneity (contaminant speciescannot be detected in the composition by conventional detectionmethods).

“Mixed population” or “complex population” refers to any samplecontaining both desired and undesired nucleic acids. As a non-limitingexample, a complex population of nucleic acids may be total genomic DNA,total RNA or a combination thereof. Moreover, a complex population ofnucleic acids may have been enriched for a given population but includesother undesirable populations. For example, a complex population ofnucleic acids may be a sample which has been enriched for desiredmessenger RNA (mRNA) sequences but still includes some undesiredribosomal RNA sequences (rRNA).

“RNA” or “transcribed RNA” as used herein, include, but are not limitedto RNA transcripts, pre-mRNA transcript(s), transcript processingintermediates, mature mRNA(s) ready for translation and transcripts ofthe gene or genes, or nucleic acids derived from the RNA transcript(s).Transcript processing may include splicing, editing and degradation.Expressed RNAs may be mRNAs that code for a protein or non-coding RNAs.Non coding RNA (ncRNA) refers to all RNAs that are not messenger RNAs(mRNA). Although most genes are transcribed into messenger RNA thatencode proteins, ncRNA genes generate transcripts lacking proteincodingpotential (e.g. Functional RNA (fRNA), Micro RNA (miRNA), Ribosomal RNA(rRNA), Small Interfering RNA (siRNA), Small Nuclear RNA (snRNA), Smallnon-mRNA RNA (snmRNA), Small nucleolar RNA (snoRNA), Small temporal RNA(stRNA), Transfer RNA (tRNA)—For review See Eddy SR, Nature ReviewsGenetics, 2:919, 2001). The ncRNAs may function at a regulatory,catalytic or structural level. Several non-coding RNA genes have beenfound to be imprinted or included in imprinted domains and implicated inregulating the imprinted expression of coding transcripts (e.g. H19,AIR, XIST, Rian, DGCR5, IPW, GNAS1, ZNF127-AS, PAR-SN, PAR-1, PAR-5,UBE3A-AS, KvDMR-1). As used herein, a nucleic acid derived from an RNAtranscript refers to a nucleic acid for whose synthesis the RNAtranscript or a subsequence thereof has ultimately served as a template.Thus, a cDNA reverse transcribed from an mRNA, a cRNA transcribed fromthat cDNA, a DNA amplified from the cDNA, an RNA transcribed from theamplified DNA, etc., are all derived from the mRNA transcript anddetection of such derived products is indicative of the presence and/orabundance of the original transcript in a sample. An RNA sampleincludes, but is not limited to, RNA transcripts of the gene or genesand any nucleic acid sample derived from the RNA, such as, cDNA reversetranscribed from the RNA, cRNA transcribed from the cDNA, DNA amplifiedfrom the RNA, RNA transcribed from amplified DNA, and the like. Likewisea genomic DNA sample includes isolated genomic DNA and any nucleic acidsample derived from the isolated genomic DNA, such as, isolated genomicDNA that has been fragmented and labeled, as well as nucleic acidsamples derived from the isolated genomic DNA, such as amplified doubleor single stranded fragments of the isolated genomic DNA, RNAtranscribed from the amplified DNA, and the like. The genomic DNA samplecontains sequence information that represents the information content ofthe genome and the RNA sample contains sequence information thatrepresents the information content of the transcribed RNA.

A “fragment”, “segment”, or “DNA segment” refers to a portion of alarger DNA polynucleotide or DNA. A polynucleotide, for example, can bebroken up, or fragmented into, a plurality of segments. Various methodsof fragmenting nucleic acid are well known in the art. These methods maybe, for example, either chemical or physical in nature. Chemicalfragmentation may include partial degradation with a DNase; partialdepurination with acid; the use of restriction enzymes; intron-encodedendonucleases; DNA-based cleavage methods, such as triplex and hybridformation methods, that rely on the specific hybridization of a nucleicacid segment to localize a cleavage agent to a specific location in thenucleic acid molecule; or other enzymes or compounds which cleave DNA atknown or unknown locations. Physical fragmentation methods may involvesubjecting the DNA to a high shear rate. High shear rates may beproduced, for example, by moving DNA through a chamber or channel withpits or spikes, or forcing the DNA sample through a restricted size flowpassage, e.g., an aperture having a cross sectional dimension in themicron or submicron scale. Other physical methods include sonication andnebulization. Combinations of physical and chemical fragmentationmethods may likewise be employed such as fragmentation by heat andion-mediated hydrolysis. See for example, Sambrook et al., “MolecularCloning: A Laboratory Manual,” 3^(rd) Ed. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, New York (2001) (“Sambrook et al.) which isincorporated herein by reference for all purposes. These methods can beoptimized to digest a nucleic acid into fragments of a selected sizerange. Useful size ranges may be from 100, 200, 400, 700 or 1000 to 500,800, 1500, 2000, 4000 or 10,000 base pairs. However, larger size rangessuch as 4000, 10,000 or 20,000 to 10,000, 20,000 or 500,000 base pairsmay also be useful.

“Restriction enzymes” recognize in general a specific nucleotidesequence of four to eight nucleotides (through this number can vary) andcut a DNA molecule at specific site. For example, the restriction enzymeEcoRI recognized the sequence GAATTC and will cut the DNA between the Gand the first A. Many different restriction enzymes can be chosen for adesired result (For description of many restriction enzymes, see, NewEngland BioLabs Catalog which is herein incorporated by reference in itsentity for all purposes). Methods for conducting restriction digestswill be known to those skilled in the art. For thorough explanation ofthe use of restriction enzymes, see for example, section 5, specificallypages 5.2 to 5.32 of Sambrook et al., incorporated by reference above.This method can be used for complexity management of nucleic acidsamples such as genomic DNA, see U.S. Pat. No. 6,361,947, which ishereby incorporated by reference in its entirety.

“In silico digestion” is a computer-aided simulation of enzymaticdigests accomplished by searching a sequence for restriction sites. Insilico digestion provides for the use of a computer system to modelenzymatic reactions in order to determine experimental conditions beforeconducting any actual experiments. An example of an experiment would beto model digestion of the human genome with specific restriction enzymesto predict the sizes of the resulting restriction fragments.

A primer is a single-stranded oligonucleotide capable of acting as apoint of initiation for template-directed DNA synthesis under suitableconditions e.g., buffer and temperature, in the presence of fourdifferent nucleoside triphosphates and an agent for polymerization, suchas, for example, DNA or RNA polymerase or reverse transcriptase. Thelength of the primer, in any given case, depends on, for example, theintended use of the primer, and generally ranges from 15 to 30nucleotides. Short primer molecules generally require coolertemperatures to form sufficiently stable hybrid complexes with thetemplate. A primer need not reflect the exact sequence of the templatebut must be sufficiently complementary to hybridize with such template.The primer site is the area of the template to which a primerhybridizes. The primer pair is a set of primers including a 5′ upstreamprimer that hybridizes with the 5′ end of the sequence to be amplifiedand a 3′ downstream primer that hybridizes with the complement of the 3′end of the sequence to be amplified.

A “genome” is all the genetic material in the chromosomes of anorganism. Genome may be multichromosomal such that the DNA is cellularlydistributed among a plurality of individual chromosomes. For example, inhuman there are 22 pairs of chromosomes plus a gender associated XX orXY pair. DNA derived from the genetic material in the chromosomes of aparticular organism is genomic DNA. A genomic library is a collection ofclones made from a set of randomly generated overlapping DNA fragmentsrepresenting the entire genome of an organism.

An “allele” refers to one specific form of a gene within a cell orwithin a population, the specific form differing from other forms of thesame gene in the sequence of at least one, and frequently more than one,variant sites within the sequence of the gene. The sequences at thesevariant sites that differ between different alleles are termed“variances”, “polymorphisms”, or “mutations”.

At each autosomal specific chromosomal location or “locus” an individualpossesses two alleles, one inherited from the father and one from themother. An individual is “heterozygous” at a locus if it has twodifferent alleles at that locus. An individual is “homozygous” at alocus if it has two identical alleles at that locus.

“Polymorphism” refers to the occurrence of two or more geneticallydetermined alternative sequences or alleles in a population. Apolymorphic marker or site is the locus at which divergence occurs.Preferred markers have at least two alleles, each occurring at frequencyof greater than 1%, and more preferably greater than 10% or 20% of aselected population. A polymorphism may comprise one or more basechanges, an insertion, a repeat, or a deletion. A polymorphic locus maybe as small as one base pair. Polymorphic markers include restrictionfragment length polymorphisms, variable number of tandem repeats(VNTR's), hypervariable regions, minisatellites, dinucleotide repeats,trinucleotide repeats, tetranucleotide repeats, simple sequence repeats,and insertion elements such as Alu. The first identified allelic form isarbitrarily designated as the reference form and other allelic forms aredesignated as alternative or variant alleles. The allelic form occurringmost frequently in a selected population is sometimes referred to as thewildtype form. Diploid organisms may be homozygous or heterozygous forallelic forms. A diallelic polymorphism has two forms. A triallelicpolymorphism has three forms. Single nucleotide polymorphisms (SNPs) areincluded in polymorphisms.

Single nucleotide polymorphism (SNPs) are positions at which twoalternative bases occur at appreciable frequency (>1%) in the humanpopulation, and are the most common type of human genetic variation. Thesite is usually preceded by and followed by highly conserved sequencesof the allele (e.g., sequences that vary in less than 1/100 or 1/1000members of the populations). A single nucleotide polymorphism usuallyarises due to substitution of one nucleotide for another at thepolymorphic site. A transition is the replacement of one purine byanother purine or one pyrimidine by another pyrimidine. A transversionis the replacement of a purine by a pyrimidine or vice versa. Singlenucleotide polymorphisms can also arise from a deletion of a nucleotideor an insertion of a nucleotide relative to a reference allele.

“Genotyping” refers to the determination of the genetic information anindividual carries at one or more positions in the genome. For example,genotyping may comprise the determination of which allele or alleles anindividual carries for a single SNP or the determination of which alleleor alleles an individual carries for a plurality of SNPs. A genotype maybe the identity of the alleles present in an individual at one or morepolymorphic sites.

“Genomic imprinting” or “allelic exclusion according to parent oforigin” is a mechanism of gene regulation by which only one of theparental copies of a gene is expressed. Paternal imprinting means thatan allele inherited from the father is not expressed in offspring.Maternal imprinting means that an allele inherited from the mother isnot expressed in offspring. Imprinted genes are the genes for which oneof the parental alleles is repressed whereas the other one istranscribed and expressed. The expression of an imprinted gene may varyin different tissues or at different developmental stages. Imprintedgenes may be expressed in a variety of tissue or cell types such asmuscle, liver, spleen, lung, central nervous system, kidney, testis,ovary, pancreas, placenta, skin, adrenal, parathyroid, bladder, breast,pituitary, intestinal, salivary gland blood cells, lymph node and otherknown in art. For instance, Igf2 imprinting results in repression of thematernally-derived allele in most tissues except brain, adult liver andchondrocytes (Vu T. H. and Hoffman A. R. (1994) Nature, 371:714-717,UBE3A (ubiquitin protein ligase 3) is paternally repressed exclusivelyin brain, KCNQ1 is paternally repressed in most tissues but is notimprinted in heart and WT1 (Wilms' tumor gene) is paternally repressedin cells of placenta and brain but not in kidney.

Genes may be imprinted only during specific developmental stages of anorganism. For example, PEG1/MEST is maternally repressed in fetal tissuebut biallelically expressed in adult blood. Also, genes may bepaternally or maternally repressed in a particular species (e.g. murineversus human, Killian K. J. et al. (2001) Hum. Mol. Genet.,10:1721-1728). Loss of imprinting or LOI is said to occur when thenormally silenced allele of an imprinted gene is activated. Both allelesof a gene that is usually imprinted may be expressed at equal levels.

Degree of allelic imbalance refers to the differential expression of thetwo alleles of a gene for which imprinting is assessed (i.e. maternalversus paternal: M/P). RNA is typically transcribed from maternal andpaternal genes equally (i.e.50/50). Imprinting is an example whereeither the maternal or paternal allele is transcribed and corresponds toa 100/0 or 0/100 expression ratio. Allelic imbalance is found when oneparent is preferentially transcribed more than the other (e.g. 80/20).

The diseases caused by imprinting, abnormal imprinting such as LOI, andmonoallelic expression include, but are not limited to, Prader-Willisyndrome, Angelman syndrome, Beckwith-Wiedmann syndrome, Silver-Russelsyndrome, cancers, sudden infant death syndrome, birth defects, mentalretardation, diabetes and gestational diabetes, neurological disorders,autism, bipolar affective disorder, epilepsy, schizophrenia, Tourettesyndrome and Turner syndrome.

The term “gene expression” refers to the process in which geneticinformation flows from DNA to functional molecule, such as protein orRNA molecules. Monoallelic expression refers to the expression of onlyone of the two alleles of the gene in a cell, because of imprinting,X-inactivation, or gene rearrangements that take place withinimmunoglobulin and T-cell receptor genes.

Analysis of monoallelic expression of genes in an individual can beperformed on a biological sample. Example of tissue or cells from whichRNA can be extracted and analyzed includes, but is not limited to, skin,ligaments, eye, kidney, liver, heart, lung, bone-marrow, neural tissue,motor neurons, sensory neurons, blood-white cells and other myelocytelineage and muscle.

The term “imprinting map” illustrates the chromosomal regions of thegenome subject to imprinting. Chromosomes that are likely to showimprinting include 2, 6, 7, 11, 14, 15, 16, 20 and X (Ledbetter D. H.and Engel E. (1995) Hum. Mol. Genet. 4: 1757; Morison I. M. and Reeve A.E. (1998) Hum. Mol. Genet. 7: 2599). Chromosome regions could be labeledaccording to the phenotype (i.e. M for maternal when no paternal alleleis expressed or P for paternal when no maternal allele is expressed).

III. The Process

a. Methods to Identify Imprinted Genes In general methods are providedfor determining if both alleles of a heterozygous gene are beingexpressed. Diploid organisms generally have two copies of each gene, butboth copies are not always expressed at equal levels and in somecircumstances only one of the copies is expressed. For many genes thisallelic imbalance is required for normal development and a deleteriousphenotype results when the imbalanced regulation is altered. Imprintedgenes provide multiple examples where it is important that only one copyof the gene be expressed. Allelic imbalance may be regulateddevelopmentally or in a tissue specific manner or it may beconstitutive. To determine if both alleles of a heterozygous gene arebeing expressed the hybridization pattern resulting from the genomicDNA.

Several methods have been used to determine imprinted genes (For reviewsee, Oakey R. J. and Beechey C. V. (2002), Trends Genet., 18:359-366;Kelsey G. and Reik W. (1998), Methods, 14:211-234). These methodsinclude subtractive hybridization (Kuroiwa Y. et al. (1996) Nat. Genet.,12:186-190; Kaneko-Ishino Y. et al. (1995), Nat. Genet., 11:52-59;Kagitani F. et al. (1997), Nucleic Acids Res., 25:3428-3432; Piras .G etal. (2000), Mol. Cel. Biol., 20:3308-3315), differential display PCR(Hagiwara Y. (1997), Proc. Natl. Acad. Sci. U.S.A, 94:9249-9254;Georgiades P. et al., Development, 127: 4719-4728; Takada S. (2000),Curr. Biol., 10:1135-1138), serial analysis of gene expression(Velculescu V. E. (1995), Science, 270:368-369; Velculescu V. E. (2000),Trends Genet., 16:423-425), microarrays (Choi J.D. (2001), Mamm. Genome,12:758-764; Kobayashi S. (2000), Genes Cells, 5:1029-1037; Mizuno Y.(2002), Biochem. Biophys. Res. Commun., 290:1499-1505), antisense andnon-coding RNAs (Lehner B. (2002), Trends. Genet., 18:63-65) and singlenucleotide polymorphisms (Coghill E. L. (2002), Nat. Genet., 30:255-256;Uejima H et al. (2000), Nat. Genet., 25: 375-376).

The methods of the present invention provide a systematic approach tostudy whole genome allelic exclusion. In some embodiments genes that areheterozygous in the genome are analyzed to determine if the RNAtranscribed from that gene comes from one allele or both alleles of thegene. Heterozygous genes that are expressed with an allelic imbalancemay be identified. Rapid, efficient and scalable methods to identifyimprinted genes are included. Genes that display errors in imprintingmay also be identified. In another embodiment arrays for carrying outthis analysis are disclosed. Imprinting and errors in imprinting may betissue specific or may be present only during specific developmentalstages. In some embodiments the method may be used to screen more than100, 1000, 5000, 10,000, 50,000 or 100,000 genes.

Both the silent and the active parental alleles of an imprinted gene maybe retained in the genome. To determine if a gene is imprinted nucleicacid samples from an individual may be characterized at both the genomicand the transcriptional levels. A gene that is imprinted may be presentin two allelic forms in the genome but only one of the alleles istranscribed, resulting in the detection of both alleles in the genomicsample but only one of the alleles in the transcribed RNA. In someembodiments SNPs, within the transcribed regions of a gene, are used todetermine if multiple alleles of a gene are present in the genome and tomonitor the expression of the different allelic forms of the gene.Microarrays designed to interrogate polymorphisms may be used toidentify the presence or absence of individual SNP alleles in genomicDNA or in transcribed RNA (see, for example, U.S. patent applicationSer. Nos. 09/916,135 and Ser. No. 10/264,945). The methods may be usedto determine if only the paternal allele is expressed, if only thematernal allele is expressed or if both alleles are expressed. Forexample, if the individual is AB, carrying both the A and B alleles, theA allele being the maternal copy and the B allele being the paternalcopy, but only the A allele is detected in the RNA then the gene may beimprinted with the paternal copy being silent and the maternal copybeing expressed. If both the A and B alleles are present in the RNA thegene displays biallelic expression and is not imprinted. If only the Ballele is present in the RNA then the maternal allele is silent and onlythe paternal allele is expressed.

The parental origin of an imprinted allele may also be determined. If anorganism is AB at a given SNP in an imprinted gene and at least one ofthe parents is homozygous the methods may be used to determine theparental origin of the expressed allele. For example, if the expressedallele is B and the mother was AA and the father BB then the silencedallele is the maternal allele, the A allele, and the expressed allele isthe paternal allele, the B allele. The parental origin of an imprintedallele may effect the penetrance and severity of diseases associatedwith imprinting.

In some embodiments both alleles are present in the RNA but one alleleis present at higher levels than the other allele. This allelicimbalance may be detected by the present methods.

Single nucleotide polymorphisms (SNPs) may be used for testing humangenetic variation. SNPs are present in the genome in a high density,they are stable mutations relative to other markers such asmicrosatellites and they may be analyzed using high throughput typingmethodologies which may be relatively inexpensive. Since SNPs are themost abundant form of human genetic variation, they are useful markersfor genomic research (Collins F. S. et al., (1996) Science,278:1580-1581; Wang D. G. et al. (1998), Science, 280:1077-1082; Gray I.C. et al. (2000), Hum. Mol. Genet., 9:2403-2408). On average, SNPsoccurs every 1,000 bases when two human chromosomes are compared (TheInternational SNP Map Working Group, Science, 409:928-933, 2001).Strategies for detection and identification of polymorphisms aredescribed in U.S. Pat. Nos. 5,858,659, 6,361,947, U.S. patentapplication Ser. Nos. 09/916,135, Ser. No. 09/920,491, Ser No.09/910,292, Ser. No. 10/264,945 and Ser. No. 10/013,598, and Dong et al.(2001), Genome Res.,11:1418-1424, each of which is incorporated byreference in their entity for all purposes. Some embodiments of thepresent methods employ pre-characterized SNPs. That is, genotyping maybe performed after the location and the nature of polymorphic forms at asite have been determined. Genotyping arrays may be designed to analyzemany different polymorphisms simultaneously, for example, an array maybe designed to interrogate 1000, 5,000, 10,000, or 20,000 to 10,000,20,000, 100,000, 500,000, or 1,000,000 different polymorphic positions.

In some embodiments a genomic DNA sample or a nucleic acid samplederived from genomic DNA is hybridized to a genotyping array and one ormore heterozygous SNPs are identified. SNPs that are heterozygous in thegenomic DNA may then be analyzed to determine if RNA transcribed fromthat region is homozygous or heterozygous. The analysis of thetranscribed RNA may be by hybridizing the transcribed RNA or a nucleicacid sample derived from the transcribed RNA to a genotyping array. Inorder to determine if one or more heterozygous SNPs are homozygous inthe transcribed RNA the RNA may be converted to cDNA and hybridized to asecond copy of the array. In some embodiments the cDNA is furtheramplified. In some embodiments the transcribed RNA is labeled directlyand hybridized directly to an array. Methods for amplification andlabeling are described above and in U.S. patent application Ser. Nos.09/285,658, Ser. No. 09/961,709, Ser. No. 10/090,320 and Ser. No.09/738,892. The gene is identified as an imprinted gene if the SNP isheterozygous in the genomic DNA but homozygous in the RNA.

In some embodiments genes that are known to be imprinted are analyzed todetect failure of imprinting or improper imprinting. For example, somegenes are normally expressed only from the maternal allele andexpression from both maternal and paternal alleles results in anabnormal or disease phenotype. If the individual is heterozygous at thatlocation the expression products from each allele may be detected byhybridization to a genotyping array. In some embodiments the amount ofRNA resulting from each allele may be determined. An approximatelyequivalent amount of expression is expected from each allele.

In some embodiments the genomic DNA and the RNA or their amplificationproducts bear different labels and are hybridized simultaneously to thesame array, meaning the same copy of an array. In some embodiments thesamples may be labeled with the same label and each sample may behybridized to a different copy of the same array. The samples may bedifferentially labeled so that the signal resulting from the genomic DNAand the signal resulting from the RNA may be distinguished (i.e. twocolor labeling). A variety of different fluorescent labels are availableand may be used. For example, one sample can be labeled with fluoresceinand the other with biotin, which can be stained withphycoerythrin-steptavidin after hybridization (See U.S. Pat. Nos.6,013,449 and 6,309,822, which are both incorporated herein by referencein their entireties).

For assay of genomic DNA, virtually any biological sample (other thanpure red blood cells) is suitable. For example, convenient tissue sampleinclude whole blood, saliva, buccal, tears, semen, urine, sweat, fecalmaterial, skin and hair. The invention analyses sequentially orsimultaneously the polymorphic forms of the RNA transcript. In general,the same probe arrays that are used for analyzing polymorphic forms ingenomic DNA can be used for analyzing polymorphic forms for transcripts,for example the Human 10K Mapping Set, (Affymetrix, Santa Clara). RNAfor analysis is isolated from a biological tissue or fluid or cells inwhich the gene of interest is expressed. Sample includes sputum, blood,blood cells (e.g., white cells), tissue or fine needle tissue samples,urine, peritoneal fluid and pleural fluid, or cells therefrom.Biological samples may also include sections of tissues such as frozensections taken from histological purposes. Methods for isolating mRNAare described in Chapter 3 of Laboratory techniques in Biochemistry andMolecular Biology: Hybridization With Nucleic Acid Probes, Part I.Theory and Nucleic Acid Preparation, P. Tijssen, ed. Elsevier, N. Y.(1993). For example total RNA is isolated from a given sample usingacid-guanidium-phenol-chloroform extraction method and polyA⁺ mRNA isisolated by oligodT column chromatography or by using (dT)_(n) magneticbeads (see, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual (2^(nd) ed.), Vol. 1-3, Cold Spring Harbor Laboratory (1989))

Methods of genotyping using a nucleic acid array following complexityreduction have been described, for example, in U.S. Pat. No. 6,361,947and U.S. patent application Ser. No. 09/916,135, also see, Dong S. etal. (2001), Genome Res., 11:1418-1424, each of which are incorporatedherein by reference in their entireties. In brief, this method comprisesfragmenting nucleic acids sample to form fragments, ligating adaptors tothe fragments and amplifying the fragments under conditions that favoramplification of fragments of a particular size. In silico digestion isused in many embodiments to predict the SNPs that will be present when agenome is digested with a particular restriction enzyme or enzymes. TheSNPs and corresponding fragment sizes can be further separated bycomputer into subsets according to fragment size. The information isthen used to design arrays to interrogate SNPs predicted to be presentin a particular size fraction resulting from a particular digestion andamplification method. Arrays may be designed to interrogate a particularsubset of sequences or fragments that may include a subset ofpolymorphisms. In some embodiments a subset of a genome is isolated by,for example, preferential amplification of a subset of fragments. Insome embodiments the subset of fragments that is preferentiallyamplified are those fragments that are of a selected size range, forexample, between 1, 200, 400, 500, or 1,000 and 800, 1,000, 2,000 or5,000 bases. In many embodiments the array is designed to interrogateone or more polymorphic positions predicted to be present in thefragments of the subset of fragments isolated or amplified. In someembodiments SNPs are amplified by target specific amplification usingone or more primers that hybridize near the SNP or interest. Acollection of SNPs may be amplified using a collection of targetspecific primers. The array may be designed to interrogate the SNPs inthe collection of SNPs.

In one embodiment, mRNA is extracted from different tissues or from thesame tissue at different developmental stages for example adult brain,adult liver, fetal brain, fetal liver. In some embodiments imprintingthat is developmentally regulated or regulated in a tissue specificmanner may be identified. Genomic DNA and mRNA are genotyped and arecompared in order to identify imprinted genes that are tissue specificor that are developmentally regulated. To confirm or detect tissue ordevelopmental regulation of imprinting, an individual whose genomic DNAis heterozygous for a SNP is identified and RNA is analyzed to determineif the gene shows monoallelic or biallelic expression in differenttissues or at different developmental stages. For example, the followingcomparisons are made: 1) fetal brain versus adult brain, 2) fetal liverversus adult liver, 3) adult brain versus adult liver, 4) fetal brainversus fetal liver. Using this method one can produce a representationof the genes imprinted in the major organs of an individual at differentdevelopmental stages. Imprinting in an individual may be compared to theimprinting expected in a normal sample.

In one embodiment, analysis of imprinting genes is performed on tissuesor cells to be used for transplantation in order to avoid thepossibility of increasing the disease risk in the transplant recipient.

Another aspect of the invention is the creation of a genome-widechromosomal imprinting map. One embodiment describes a method ofidentifying imprinted genes by screening a large number of genes. In oneembodiment, gene(s) within 2 to 4 million base pairs of a knownimprinted gene is identified and assayed whether it is uniparentallyexpressed or not. Imprinted genes are indeed often clustered in largedomains. For instance, studies have identified a set of novel imprintedgenes within 1.5 Mbases of chromosome 15q (implicated in Prader-Willisyndrome and Angelman syndrome. (See, Lee S. and Wevrick R. (2000) Am.J. Hum. Genet,. 66:848-858). In one embodiment a human chromosomalimprinting map is established. This map has important clinicalimplications, particularly in the area of prenatal diagnosis. In oneembodiment, it is determined if these newly identified imprinted genesare associated with a disease state. One embodiment describes arraysdisplaying oligonucleotides probes for interrogating genes imprinted indifferent tissues and/or at different developmental stages. In anotherembodiment, oligonucleotide arrays are used to interrogate imprintedgenes in different species.

b. Correlation of Imprinted Genes With Phenotypic Characteristics

In some embodiments errors in imprinting may be correlated withphenotype. Correlation may be used to assess risk for disease, predictresponse to environment, predict treatment outcome or to determine theimpact of cloning on imprinting.

Imprinted genes that correlate with disease are particularly interestingbecause they represent mechanisms to accurately diagnose syndromesrelated to genomic imprinting. Several genetic changes are known todisrupt the expression of imprinted genes. Large deletions on thechromosome containing the active allele can disrupt or delete theexpressed copy of an imprinted gene causing loss of function.Uniparental disomy, a duplication of one allele with loss of the other,may result in loss of function if the inactive allele is duplicated.

A significant proportion of imprinted genes have been shown to beimplicated in the control of fetal growth. Some diseases such asPrader-Willi syndrome (Nicholls R. D. and Knepper J. L. (2001), Annu.Rev. Genomics Hum. Genet., 2:153-175), Angelman syndrome (Rougelle C. etal. (1997), Nat. Genet., 17 :14-15), Silver-Russel syndrome (Hitchins M.P. et al. (2001), Eur. J. Hum. Genet., 9:82-90) and Beckwith-Wiedmannsyndrome have been correlated with imprinting mechanism (for review see,Falls G. J. (1999) Am. J. Path., 154:635-647). Parent-of-origininheritance effects, i.e. occurrence or severity of the symptoms,suggest that imprinted genes are also implicated in autism, bipolaraffective disorder, epilepsy, schizophrenia, Tourette syndrome andTurner syndrome. Also, it has been shown that some tumors are linkedwith the preferential loss of a particular parental chromosome,indicating the involvement of imprinted genes. Imprinted genesimplicated in human carcinogenesis include Igf2, WT1, p57^(KIP2), p73,NOEY2 and M6P/Igf2R (for review, see Fall G. J. et al. (1999), Am. J.Path., 154:635-647). Loss of imprinting (LOI) in cancer can lead toactivation of normally silent alleles of growth-promoting genes (Rainieret al. (1993) Nature 362: 747). This phenomenon has been observed invarious adult cancers including lung, breast, gastrointestinal,esophageal, endometrial, uterine, ovarian, cervical, skin, endocrine,urinary, prostate, colorectal cancers and in leukemia and lymphoma (Forreview, see Jirtle R. L. (1999) Exp. Cell Res., 248:18-24). LOI may beused to diagnose or predict risk of certain diseases such as coloncancer. In some embodiments LOI is monitored in response to treatment todetermine if LOI is impacted or reversed. Monitoring of LOI may be usedto evaluate candidate drugs or treatments for ability to reverse LOI.

In one embodiment, individuals are tested for the presence or theabsence of an imprinted gene and for the phenotypic trait or traits ofinterest. The presence or absence of imprinting in individuals thatexhibit a particular phenotype is compared to imprinting of the samegene in individuals who lack the phenotype to determine if the presenceor absence of imprinting in a particular gene or genes is associatedwith the phenotype.

In another embodiment the methods of the invention provide a method todetermine whether the imprint status at a particular locus ispolymorphic. Some imprinted genes such as Igf2, WT1, HTR2A appear to bepolymorphic, the gene being imprinted in some individuals but not inothers or imprinted in humans but not in other species such as in mouse(Killian K. J. et al. (2001) Hum. Mol. Genet., 10:1721-1728). It is notknown yet if polymorphic imprinting can determine individual and/orspecies differences in susceptibility to disease.

In some embodiments, the methods of the invention may be used toevaluate the environmental influence of physical, chemical or radiationagents on the imprinting status of the genome and assess how it isrelated to known diseases or phenotypes. Since DNA methylation andchromatin structure are important in regulation of genomic imprinting,environmental factors capable of causing epigenetic changes in DNA canpotentially alter the expression of imprinted genes resulting in geneticdiseases including cancer and behavioral disorders (Murphy S. K. andJirtle R. L. (2000) Environ. Health Perspect., 108 (Suppl.1): 5-11). Inanother embodiment the methods may be used to correlate imprinting withexposure of an individual or an individual's parents to a stress, suchas a chemical mutagen. For example, a correlation has been found betweenparental exposure to chemical mutagens and the occurrence ofPrader-Willi syndrome in children (Cassidy S. B. et al. (1989) Am. J.Hum. Genet., 44:806-810).

Cloning of various mammalian organisms has been achieved by nucleartransfer technology: a nucleus is removed from a somatic donor cell andis transplanted in an enucleated oocyte (for review see, Gurdon J. B.and Colman A. (1999) Nature, 402:743-746). The renucleated oocyte willcarry the genome of only the donor individual and if implanted willdevelop to a cloned individual genetically similar to the nuclear donor.Cloning in mammals is very inefficient since less than 1% of the clonessurvive to term. Most of those who survive develop severe abnormalities(such as fetal overgrowth, enlarged heart, placental overgrowth,defective kidneys, immature lung development, reduced immunity todisease . . . ) and die soon thereafter (Young L. E. et al. (1998) Rev.Reprod., 3:155-163). Fetal over growth or large offspring syndrome is amajor problem encountered in cloning and is believed to result fromparental genomic imprinting (Young L. E. et al. (2001) Nat. Genet.,27:153-154). Imprinted genes are switched off in the embryo and many ofthem belong to the fetal growth pathway. For instance, paternalimprinted genes tend to enhance fetal growth (such as Igf2) whereasmaternal imprinted genes (such as H19) tend to suppress fetal growth.The genome-wide balance in the dosage of theses factors determines thesize of the offspring.

Imprinting poses a potential problem for cloning by nuclear transfer. Tobe successful in directing development, an adult nucleus would have tohave maintained a stable imprinting pattern and this pattern would needto be preserved or replaced following nuclear transfer. The success ofproducing live-born animals by this procedure suggests that such issuesare not insurmountable, but there may be imprinting errors thatcontribute to the high failure rate seen in cloning experiments to date.In some embodiments of the present invention methods are provided fordetermining if a cloned individual has a normal imprinting pattern forone or more genes. The methods may also be used to identify genes thatshow imprinting during one stage of development but not during anotherstage of development.

In some embodiments, imprinted gene expression patterns are comparedbetween clones and normal embryos. It has been shown that the level ofexpression of imprinted genes such as Igf2R can vary significantly incloned embryos that are cultured in vitro before implantation (Young L.E. et al. (2001) Nat. Genet., 27:153-154). Preimplantation cultureconditions can influence the regulation of growth-related imprintedgenes (Khosla S. et al. (2001) Biol. Reprod., 64:918-926). Loss ofimprinting during embryo culture could also account to the reduced birthweight of progeny resulting from human in vitro fertilization. Moreover,two children conceived by intracytoplasmic sperm injection have beenshown to develop Angelman syndrome due to a sporadic imprinting defect(Cox G. F. et al. (2002) Am. J. Hum. Genet., 71:162-164). In order toobtain and improve the success rate of obtaining viable and healthyprogeny using cloning methods, it is essential to examine and evaluatethe expression of the imprinted genes before implantation in the uterus.In some embodiments, expression of imprinted genes is assayed in embryosat different key stages of development prior to implantation in theuterus. In another embodiment preimplantation embryos are diagnosed forany known disease correlated with imprinted genes. In another embodimenta cloned tissue or organism is assessed for imprinting of one or moregenes.

In some embodiments the methods are used to determine the imprintingstatus of genes in cells derived from stem cells prior to use of thesecells for cloning or prior to transplantation of tissue derived fromembryonic stem cells into a patient. Stem cells such as embryonic germcells (EG cells) or embryonic stem cells (ES cells) are pluripotentcells that can differentiate into any cell type. Stem cell derivedtissues have the potential to be used for transplantation therapy toreplace damaged differentiated cells or cells lost in many disorderssuch as diabetes mellitus (to replace insulin-secreting beta cells ofthe pancreas) and Parkinson's disease (to replace dopamine secretingcells of the brain). However, work on embryonic stem cells and tissuesderived from stem cells indicates that these methods may introduceimprinting errors that might result in a disease phenotype in theindividual. From this perspective, it is important to be able to checkthe expression of imprinted genes in stem cell derived tissues beforetransplantation.

Errors in silencing of genes on the X-chromosome may also be detected bycomparing the genotype of multiallelic loci and the expressed RNA.X-chromosome inactivation is random so in some cells the maternal copywill be inactivated while in other cells the paternal copy will beinactivated. The methods may be used to determine which copy of theX-chromosome is inactivated in a given cell by comparing the RNAexpressed in the cell with the genotype of the maternal and paternal Xchromosomes for at least one heterozygous gene.

EXAMPLES

Reference will be now made in detail to illustrative embodiments of theinvention. While the invention will be described in conjunction with theillustrative embodiments, it will be understood that the invention isnot limited. On the contrary, the invention is intended to coveralternatives, modifications and equivalents, which may be includedwithin the spirit and scope of the invention. The following example isintended to illustrate the invention.

Example 1 Identification of Imprinted Genes in an Individual

A total of 5 μg of human genomic DNA is digested with restrictionenzyme, in 1× restriction enzyme buffer for 2 hours at 37° C. Heatingthe reaction at 70° C. for 20 minutes inactivates restriction enzyme.The digestion fragments are then ligated with adaptors with 8 mM DTT, 1mM ATP and 5000 units T4 DNA ligase at 16° C. for 2 hours. The reactionis stopped by heating the mixture for 10 minutes at 70° C. Fragments arethen amplified by PCR with 5 μM of the appropriate primers in 250 μMdNTPs, 15 mM Tris-HCl pH 8.0, 2 mM MgCl2 and 5 units of TaqGoldpolymerase. An aliquot of the PCR product is analyzed on an agarose gelto confirm that the products have the correct average size. The PCRfragments are segmented with DNase I by incubation for 20 minutes at 37°C. and then 10 minutes at 95° C. DNA is labeled by mixing the fragmentedDNA with biotin-N⁶-ddATP and terminal transferase for 2 hours at 37° C.DNA fragments are heat denatured by boiling for 30 minutes. Denaturedenzymes are removed by centrifugation. Standard procedures are used forhybridization, washing, scanning and data analysis. PCR products arehybridized to an array designed to detect the presence or the absenceand the heterozygosity status of a given SNP containing target in thesample. After hybridization, the array is washed and stained withStreptavidin-R-phycoerythrin conjugate and washed on a fluidics station(Affymetrix). Anti-Streptavidin antibody is then added and the array isstained again with the staining solution followed by washing as in theprevious step. The arrays are then scanned with a chip scanner at 570nm. A first hybridization pattern is obtained. Single nucleotidepolymorphisms that are heterozygous in the genomic DNA are identified.

Total RNA is isolated from a specific tissue at a specific developmentalstage. Total mRNA is purified by oligo(dT) column chromatography or byusing (dT) magnetic beads. Poly(A)+mRNA is generated and reversetranscribed with a reverse transcriptase (Superscript II system, LifeTechnologies, Rockville, Md.). RNA is removed by using RNase H and RNaseA for 10 minutes at 37° C. The cDNA is purified using the Quiaquick PCRpurification kit from Quiagen (Valencia, Calif.). Before hybridizationthe cDNA is fragmented using a partial DNase I digest or by incubatingRNA in RNase free RNA fragmentation buffer (200 mM Tris-acetate pH 8.1;500 mM potassium acetate, 150 mM Magnesium acetate) and heated at 94° C.for 35 minutes and then chilled on ice. The fragmentation is confirmedby electrophoresis on agarose gel to verify the average size of thefragments. The fragmented, end-labeled cDNA is heat denatured beforebeing on an identical array than the one used to screen the genomic DNA.After hybridization and staining, the array is scanned and secondhybridization pattern is obtained. Single nucleotide polymorphisms thatare homozygous in the RNA but heterozygous in the genomic DNA areidentified. These polymorphisms establish which genes are imprinted inan individual.

Example 2 Genotyping RNA

Five Lymphoblast cell lines from Coriell Institute were cultured andharvested. Total RNA was extracted from the cell lines followed by cDNAsynthesis. After double stranded cDNA was obtained, cRNA was generated.The cRNA was labeled by incorporating biotinylated ribonucleotides intothe cRNA during synthesis. The cRNA was then fragmented andhybridization onto the “TSC_(—)0101_(—)501 and 502” genotyping arrays.There are 4658 EcoRI SNPs tiled on the p502 array and 7398 Xbal SNPstiled on the p501 array.

The cRNA was fragmented by adding 5× fragmentation buffer that containsMg²⁺ to break the phosphate bond and incubating at 94° C. for 35minutes. Fragmented cRNA was mixed into expression or WGA hybridizationcocktail. Expression hybridization cocktail contains (finalconcentration of): 0.05 μg/μl fragmented cRNA; 50 pM of controloligonucleotide B2; 0.1 mg/ml herring sperm DNA; 0.5 mg/ml acetylatedBSA and 1× MES hybridization buffer. WGA hybridization cocktail contains(final concentration of): 0.05 μg/μl fragmented cRNA; 2.75 M TMA; 0.05 MMES hybridization buffer; 5% DMSO; 5 mM EDTA; 2.5× Denhardt's; 100 pMcontrol oligonucleotide B2; 0.1 mg/ml herring sperm DNA; 0.01 mg/mlhuman cot-1 DNA and 0.01% tween-20.

After hybridization, arrays were washed, stained and scanned underexpression or WGA conditions accordingly (for expression conditions seeGeneChip® Expression Technical Manual, Affymetrix, Santa Clara, Calif.and for WGA conditions see U.S. patent Application Ser. Nos. 10/264,945and 60/417,190. Data from each cell line came from four hybridizedarrays: two p501 arrays (one expression, one WGA) and two p502 arrays.

DNAs from the same five cell lines were purchased from CoriellInstitute. WGA-FSP assay was carried out to amplify 400-800 by fragmentfrom Xb al or EcoR1 cut whole genomic DNA. The PCR products were thenfragmented, labeled and hybridized onto TSC_(—)0101_(—)501 (XbaI) arrayor onto TSC_(—)0101_(—)502 (EcoRI) array and A/A+B was plotted where Ais the signal corresponding to one allele and B is the signalcorresponding to a second allele. For the genomic DNA samples signalfrom the sense strand, RAS1 was plotted against signal from theantisense strand RAS2. For the RNA only the sense signal, RAS1, could beanalyzed.

Alternatively, double stranded cDNA made from the RNA may be hybridizedto the array. This allows detection of both the sense and antisensesignal as for the genomic DNA.

The results may be plotted on a graph of antisense signal versus sensesignal. Genotypes may be represented as relative allele signals (RAS) oneither one or both strands. RAS values are calculated from signalsobtained for the A and B alleles for each SNP according to the formulaA/A+B. An individual with an AA genotype would have a RAN value close to1, an individual with a BB genotype will have a RAS value close to 0 andan individual with an AB phenotype will have a RAS value close to 0.5.Plotting RAS1 versus RAS2 should result in a diagonal with BB genotypesnear (0,0), AB near (0.5, 0.5) and AA near (1,1). For a non-imprintedgene the genotypes of the SNP in the RNA should be similar to thegenotypes of the SNP in the DNA. In some circumstances only RAS1 will beanalyzed because RAS2 is the signal resulting from the opposite strandwhich isn't present in the RNA. The RNA may be converted to dsDNA andvalues for both RAS1 and RAS2 may be obtained. If the SNP is in animprinted gene the RAS value for the RNA in a heterozygous individualwill be close to either 1 or 0 (AA or BB) while the RAS values for theDNA will be close to 0.5 (AB).

The results of the example demonstrate that genotype information fromthe DNA is preserved in the RNA and also demonstrates the capability ofgenotyping RNA using genotyping arrays. In one of the individuals theDNA was found to be homozygous BB (RAS values close to 0) and the singlecorresponding RNA is also homozygous BB. In two individuals, the DNA isheterozygous AB (RAS values close to 0.5) and the corresponding RNA isalso heterozygous AB (RAS1 value close to 0.5) indicating no imprinting.In one individual, the DNA is homozygous AA (RAS values close to 1) andthe corresponding RNA is also homozygous AA (RAS1 value close to 1).

CONCLUSION

All publications and patent applications cited above are incorporated byreference in their entirety for all purposes to the same extent as ifeach individual publication or patent application were specifically andindividually indicated to be so incorporated by reference. Although thepresent invention has been described in some detail by way ofillustration and example for purposes of clarity and understanding, itwill be apparent that certain changes and modifications may be practicedwithin the scope of the appended claims.

1-40. (canceled)
 41. A method of Identifying at least one heterozygousgene showing monoallelic expression in an individual comprising assayinga genomic DNA sample from an individual to identify alleles of aheterozygous gene including a polymorphism, assaying an KNA sample fromthe individual to determine whether RNA is transcribed from one or moreof the alleles, whereby the heterozygous gene exhibits monoallelicexpression when RNA is expressed from only one of the alleles.
 42. Themethod of claim 41 wherein the at least one heterozygous gene showingmonoallelic expression is an imprinted gene.
 43. The method of claim 42wherein parental origin of the expressed allele of the imprinted gene isdetermined by determining which alleles of the gene are present in themother, determining which alleles of the gene are present in the father,and matching the expressed allele to one of the alleles present in themother or the father.
 44. The method of claim 41 wherein the geneshowing monoallelic expression is a gene encoding a lytnphoid-specificfactor.
 45. The method of claim 41 wherein the gene showing monoallelicexpression is a subunit of an olfactory receptor.
 46. The method ofclaim 41 wherein the gene showing monoallelic expression is a subunit ofa T cell receptor.
 47. The method of claim 41 wherein the gene showingmonoallelic expression is a subunit of an immunoglobulin.
 48. The methodof claim 41 wherein the genomic DNA sample and the RNA sample aredifferentially labeled.
 49. The method of claim 41 wherein thepolymorphism is associated with a phenotype.
 50. The method of claim 41wherein the polymorphism is associated with a disease.
 51. The method ofclaim 50 wherein the disease is cancer.
 52. The method of claim 50wherein the disease is a neurological disorder.
 53. The method of claim41 wherein the polymorphism is a single nucleotide polymorphism.
 54. Themethod of claim 41 wherein the polymorphism comprises a plurality ofsingle nucleotide polymorphisms.
 55. The method of claim 54 wherein theplurality of single nucleolide polymorphisms includes at least 1,000different polymorphic positions.
 56. The method of claim 54 wherein theplurality of single nucleotide polymorphisms includes at least 10,000different polymorphic positions.
 57. The method of claim 54 wherein theplurality of single nucleotide polymorphisms includes at least 100,000different polymorphic positions.